Methods of treating a subject for pain

ABSTRACT

The disclosure pertains to methods of treating pain. In certain embodiments, the methods comprise administering to a subject a Plasma Protein Fraction (PPF) in an amount that is effective to treat pain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.16/659,000 filed Oct. 21, 2019, which application, pursuant to 35 U.S.C.§ 119 (e), claims priority to the filing dates of U.S. ProvisionalPatent Application No. 62/751,448 filed Oct. 26, 2018 and U.S.Provisional Patent Application No. 62/842,403 filed May 2, 2019; thedisclosures of which applications are herein incorporated by reference.

FIELD

This invention pertains to the prevention and treatment of disease andaging-associated disease. The invention relates to the use of bloodproducts, such as blood plasma and blood plasma fractions to improve andaccelerate recovery from surgery, including conditions and indicationsrelated to surgery. The invention also relates to the use of bloodproducts, such as blood plasma and blood plasma fractions to alleviatechronic pain or neuropathy and to treat indications related to woundhealing.

BACKGROUND

The following is offered as background information only and is notadmitted as prior art to the present invention.

Surgery is often associated with complications from pain,cardiopulmonary issues, infections, thromboembolic issues, andpostsurgical wound healing. Additionally, it takes time for wounds toheal whether incurred from surgery itself (e.g. incisions) or incurredby accident, force, or disease and subsequently treated by a surgicalprocedure. Such complications are often further exacerbated by age.Additional complications may arise from the surgical stress responsewith subsequent demand on organ function, which are often mediated bytrauma-induced endocrine metabolic changes and activation of cascades(cytokines, complement, arachidonic acid metabolites, nitric oxide, andfree oxygen radicals). (Kehlet H., et al., Br. J. Anaesthesia, 78:606-17(1997)). During surgical stress response, the sympathetic nervous systemis activated. (Starkweather A, et al., Topics in Pain Management,32(8):1-11 (2017)). There is an increase in pituitary hormone secretion,resulting in mobilization of energy through catabolism. This in turnresults in salt and water retention. Adrenocorticotropic hormone (ACTH)secretion is increased, which results in an increase of norepinephrineand sympathetic activity. This causes cardiovascular responses such astachycardia and hypertension and glucagon is released resulting inhyperglycemia. An increase in growth hormone and cortisol also resultsin inhibition of monocyte to macrophage differentiation. This in turn,interferes with T-cell signaling/histamine production and decreasesimmune cell migration. (Id.)

Current treatment for postsurgical recovery includes reduction ofpostoperative pain as well as multimodal interventions. (Id.) Painmanagement is important in many types of surgical recoveries, and acutepain is expected. (Pinto P R, J Pain Res, 10:1087-98 (2017)).Postoperative pain is associated to a greater degree with patients whoundergo general surgery. (Couceiro T C, Rev Bras Anestesiol,59(3):314-20 (2009)). Pain also plays a negative role on clinicaloutcome because it impairs healing and recovery. Id. Replacement of thehip and knee joints is particularly associated with pain, both chronic(from, e.g. osteoarthritis) and acute. Id. Analgesics are thereforecommonly used in postoperative recovery, both during in-patientprocedures and home recovery.

One type of multimodal intervention is Enhanced Recovery After Surgery(ERAS). (Starkweather A, supra). ERAS focuses on a wide spectrum ofsurgeries, for example, colorectal surgery, orthopedics, gynecology,urology, head and neck cancer, bladder cancer, liver disease,rectal/pelvic disease, colonic pathologies, pancreative duodenectomy,gastrectomy, and bariatric and gynecologic-oncology surgery. Id. As amultimodal strategy, it emphasizes: pre-operative techniques(counseling, fluid/carbohydrate loading; shorter period of fasting);perioperative techniques (short-acting anesthetics; normothermia;antibiotic prophylaxis; thromboembolic prophylaxis; prevention ofsalt/water overload; vomiting prevention); and postoperative techniques(early oral diet; exercise; non-opioid analgesia; and post-dischargesupport). Id.

Current therapies however have failed to eliminate postoperativemorbidity and mortality. Multimodal techniques by their very nature aretime and resource consuming. And there has not been any single techniqueor pharmaceutical treatment that can match such multimodal therapy.Because of these shortfalls, there is a need for new treatments forimproving postoperative recovery.

SUMMARY

The present invention is based on the production and use of bloodproducts for treating symptoms and conditions impacting surgicalrecovery including, for example, pain and wound healing. The presentinvention recognizes, among other things, the need for new therapies forthe treatment of unwanted conditions associated with postoperativerecovery, and for improving such recovery. Derived from blood and bloodplasma, the present compositions of the invention relate to a solutionfor the failures and shortcomings of current therapies throughutilization of blood plasma fractions exhibiting efficacy in thetreatment of unwanted conditions associated with postsurgical recoveryand for improving such recovery.

The present invention also is based on the production and use of bloodproducts for treating symptoms and conditions associated with acute andchronic pain. The present invention recognizes, among other things, theneed for new therapies for alleviating pain. Although therapeutics existfor treating acute and chronic pain, many such therapies such as opioidanalgesics present a high incidence of addiction, abuse, and associatedmorbidity and mortality.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a chronic constrictive injury (CCI) experiment.Twenty-three-month-old wild type mice were administered a CCI or shamsurgery via ligation 24 hours prior to administration of a7-consecutive-day pulse dosing regimen of either PPF1, Gabapentin,recombinant human albumin (rhAlb) or vehicle control. Behavior wasassessed during weeks two through five, and tissue collection forhistology occurred during week five.

FIG. 2 is a representation depicting the location of the CCIadministered to twenty-two-month-old wild type mice. The ligation wasadministered on the sciatic nerve as indicated by the figure. The figurewas adapted from Suter M R, et al., Anesthesiology Res and Practice,(2011), which is incorporated herein by reference in its entirety.

FIG. 3 reports data from a mechanical von Frey allodynia test inwild-type mice treated with CCI or sham surgery described in FIG. 1 .Useful for the analysis of pain behavior, the hind paw enervated by thesubject sciatic nerve, was administered with von Frey filamentstimulation. The pressure at which the mouse withdrew its hind paw wasmeasured and plotted in FIG. 3 . The figure shows that mice treated withPPF1 after CCI exhibited significantly less pain (could withstand morepressure) than those treated with vehicle control after CCI. And shamoperations treated with vehicle also exhibited significantly less painthat those treated with vehicle control after CCI. This shows that PPF1has a positive effect on mechanical nociception deficits.

FIG. 4 reports data from hippocampal histology performed on the wildtype mice described in FIG. 1 . Neurogenesis was measured using thedoublecortin (DCX) marker. Mice given CCI who were treated with PPF1 hadsignificantly more neurogenesis in the hippocampus than those whoreceived vehicle. Mice given sham operations plus vehicle trendedtowards greater neurogenesis than mice given CCI and vehiclepost-surgery. Thus, PPF1 exhibited the ability to restore neurogenesisafter chronic nerve injury.

FIG. 5 reports data from hippocampal histology performed on the wildtype mice described in FIG. 1 . CD68 expression was quantified, and micegiven a CCI plus vehicle expressed a significantly greater number ofCD68 positive cells in the hippocampus than those given a CCI plus PPF1.A similar degree of difference was observed between mice given a CCIplus vehicle and those given a sham surgery plus vehicle. This showsthat PPF1 can help to block neuroinflammation resulting from chronicnerve injury.

FIG. 6 reports data from a mechanical von Frey allodynia test intwenty-two-month-old C57BL/6J mice which received CCI or sham surgeryand tested in the timeline as described in FIG. 1 . The pressure atwhich the mouse withdrew its hind paw was assessed and represented inFIG. 6 as weeks post CCI or sham surgery. The figure illustrates thatmice administered PPF1 following CCI surgery had significantly increasedtolerance to mechanical nociception at all assessed timepoints thanthose treated with vehicle after CCI. Conversely, mice administeredGabapentin only show significant improvement in mechanical nociceptionat 2 weeks following CCI surgery and are similar to vehicle treated miceat all other timepoints. Sham surgery mice show significantly increasedresponse to mechanical nociception at 3 and 5 weeks following surgicalmanipulation. Together, these data illustrate that PPF1 amelioratesperipheral pain for a greater amount of time than that of standard ofcare treatments (Gabapentin).

FIG. 7 reports data from a hot plate test on twenty-two-month-oldwild-type mice which received CCI or sham surgery and tested in thetimeline as described in FIG. 1 . This assay was performed as describedby Woolfe and Macdonald. (Woolfe G. and Macdonald A D, J. Pharmacol.Exp. Ther. 80:300-07 (1944), which is incorporated by reference hereinin its entirety). The hot plate is set to a temperature of 55° C. Miceare acclimated to being placed inside a clear cylinder for 30 minutes.The cylinder is placed upon the hot plate and a timer started. Whennocifensive behaviors (e.g. hind paw licking or jumping) are firstobserved, the time is recorded as latency. FIG. 7 illustrates hot platenocifensive latency 5 weeks after CCI or sham surgery. PPF1 treatmentare significantly less sensitive to hot plate stimuli compared to micegiven CCI plus vehicle control, indicating a rescue effect by PPF1.Whereas, standard of care effects (Gabapentin) are similar to that ofvehicle.

FIG. 8 reports data from a hot plate test on wild-type mice whichreceived CCI or sham surgery and tested in the timeline as described inFIG. 1 . FIG. 8 illustrates hot plate nocifensive latency 5 weeks afterCCI or sham surgery. PPF1 treatment and rhALB are significantly lesssensitive to hot plate stimuli compared to mice given CCI plus vehiclecontrol.

FIG. 9 reports data from a mechanical von Frey allodynia test inC57BL/6J mice which received CCI or sham surgery and tested in thetimeline as described in FIG. 1 . FIG. 9 illustrates that miceadministered PPF1 following CCI surgery had significantly increasedtolerance to mechanical nociception at all assessed timepoints thanthose treated with vehicle after CCI. Conversely, mice administeredrhALB have similar response to mechanical allodynia to vehicle treatedmice at all timepoints.

FIG. 10 reports data from sciatic nerve histological analysis(approximately 1000 μm distal from the last ligature) of myelin basicprotein (MBP) expression in C57BL/6J mice which received CCI or shamsurgery and analyzed following tissue collection after day 35 asdescribed in FIG. 1 . FIG. 10 illustrates that mice administered PPF1following CCI surgery had significantly increased MBP intensity,indicative of increased myelin expression as compared to vehicle treatedanimals. Sham mice also express increased MBP as compared to CCI injuredvehicle mice.

FIG. 11 reports data from sciatic nerve histological analysis(approximately 1000 um distal from the last ligature) of S-100 protein(expressed by Schwann cells) in C57BL/6J mice which received CCI or shamsurgery and analyzed following tissue collection after day 35 asdescribed in FIG. 1 . FIG. 11 illustrates that mice administered PPF1following CCI surgery had significantly increased S-100 intensity,indicative of increased Schwann cells (which are myelin producing cellsin peripheral nerves) as compared to vehicle treated animals. Sham micealso express increased S-100 as compared to CCI injured vehicle mice.

FIG. 12 are images selected from sciatic nerve histological analysiswhich identify the location used for quantification in FIGS. 10 and 11(approximately 1000 μm distal from the last ligature) and representativeintensities of S-100 protein (expressed by Schwann cells) and MyelinBasic Protein in C57BL/6J mice which received CCI surgery and weretreated with either vehicle or PPF1 and used for qualitative analysis ofsciatic nerve tissue after day 35 as described in FIG. 1 .

FIG. 13 reports data from spinal cord histological analysis (performedon spinal cord tissue collected from the lumbar section L4-L6) ofC57BL/6J mice which received CCI or sham surgery and analyzed followingtissue collection after day 35 as described in FIG. 1 . FIG. 13illustrates that mice administered PPF1 following CCI surgery hadsignificantly decreased BDNF intensity within the dorsal horns of thespinal cord, indicative of decreased activation of microglia within thespinal cord. As BDNF is a pro-inflammatory cytokine released byactivated microglia, these findings suggest that PPF1 is decreasing afundamental regulator of pain states within the spinal cord, normalizingthe level to that of sham (non-CCI injured) mice.

FIG. 14 reports data from spinal cord histological analysis (performedon spinal cord tissue collected from the lumbar section L4-L6) ofC57BL/6J mice which received CCI or sham surgery and analyzed followingtissue collection after day 35 as described in FIG. 1 . FIG. 14illustrates that mice administered PPF1 following CCI surgery hadsignificantly decreased CD68 intensity within the dorsal horns of thespinal cord, indicative of decreased activation of microglia within thespinal cord. As CD68 protein is expressed by activated microglia, thissuggests that PPF1 is decreasing the activation of the fundamental celltype responsible for induction of pain states within the spinal cord,normalizing the level to that of sham (non-CCI injured) mice. Datapresented in FIG. 13 and FIG. 14 indicate that PPF1 is centrallyregulating the pain state resulting from sciatic nerve injury andameliorating or preventing the establishment of pain signaling betweenthe peripheral nerves and the brain, also described as centralsensitization.

FIG. 15 are images selected from spinal cord histological analysis whichidentify the location of dorsal horns used for quantification in FIG. 14(performed on spinal cord tissue collected from the lumbar sectionL4-L6) and representative intensities of CD68 protein (expressed byactivated microglia) in C57BL/6J mice which received CCI surgery andwere treated with either vehicle or PPF1 and used for qualitativeanalysis of spinal cord tissue after day 35 as described in FIG. 1 .

FIG. 16 are images selected from spinal cord histological analysis whichidentify the location of dorsal horns used for quantification in FIG. 13(performed on spinal cord tissue collected from the lumbar sectionL4-L6) and representative intensities of BDNF protein (a cytokinereleased by activated microglia) in C57BL/6J mice which received CCIsurgery and were treated with either vehicle or PPF1 and used forqualitative analysis of spinal cord tissue after day 35 as described inFIG. 1 .

FIG. 17 depicts a chronic constrictive injury (CCI) experiment.Twenty-two-month-old wild type mice were administered a CCI or shamsurgery via ligation 2 weeks prior to administration of a7-consecutive-day pulse dosing regimen of either PPF1, rhALB or vehiclecontrol. Behavior was assessed weekly during weeks two through seven,and tissue collection for histology occurred during week seven.

FIG. 18 reports data from a mechanical von Frey allodynia test inC57BL/6J mice which received CCI or sham surgery and tested in thetimeline as described in FIG. 17 . FIG. 18 illustrates that miceadministered PPF1 two weeks following CCI surgery had significantlyincreased tolerance to mechanical nociception beginning at a timepointone week following the cessation of PPF1 treatment which was maintainedthroughout the duration of the study. Findings in FIG. 18 suggest thatPPF1 treatment initiates processes which reduce sensitivity tomechanical allodynia in a longitudinal fashion, as improved toleranceisn't evidenced until a week following treatment (in contrast withtherapies which exclusively provide benefit during treatment, such asopioid analgesics) and is sustained for at least 28 days. Conversely,mice administered rhALB have similar response to mechanical allodynia tovehicle treated mice at all timepoints.

FIG. 19 reports data from a hot plate test on wild-type mice whichreceived CCI or sham surgery and tested in the timeline as described inFIG. 17 . FIG. 19 illustrates hot plate nocifensive latency 5 weeksafter CCI or sham surgery. PPF1 treatment is significantly lesssensitive to hot plate stimuli compared to mice given CCI plus vehiclecontrol.

FIG. 20 reports data from a hot plate test on wild-type mice whichreceived CCI or sham surgery and tested in the timeline as described inFIG. 17 . FIG. 20 illustrates hot plate nocifensive latency 7 weeksafter CCI or sham surgery. PPF1 treatment is significantly lesssensitive to hot plate stimuli compared to mice given CCI plus vehiclecontrol.

FIGS. 21A and 21B provide a histological comparison between a diabeticwound (B6.BKS(D)-Lepr^(db)/J diabetic mouse model) that was untreated(FIG. 21A) or with PPF1 (FIG. 21B). Black bars indicate wound bedthickness (epidermal plus granulation layer). Arrows indicate woundboundaries. Wound bed thickness was increased in PPF1-treated mice asdetermined by wound bed thickness. PPF1 therefore demonstrates improvedwound healing.

FIGS. 22A and 22B is a histological comparison between a diabetic wound(B6.BKS(D)-Lepr^(db)/J diabetic mouse model) that was untreated (FIG.22A) or with PPF1 (FIG. 22B). Black bars indicate the granulation layer.Blue bars indicate the epidermal layer. The PPF1-treated wound exhibiteda thicker epidermal layer than the untreated wound, however thegranulation layer exhibited an even greater trend in the differencebetween PPF1-treated and untreated wounds (i.e. the granulation layerwas thicker in the PPF1-treated wounds than the untreated wounds).

FIG. 23 depicts the general design of the diabetic wound healingexperiment used in FIGS. 24 through 28 . Blood drops indicate when bloodwas collected to measure fasting glucose level. On Day 2, the skin woundwas made, and on Days 1-7 intravenous (iv) dosing was performed.Histology (signified by the microscope) was performed after sacrifice.

FIG. 24 reports the percentage of the wound still open at several timepoints post-wounding in a first study (Study 1). Mice were treated witheither PPF1 (150 μL) for 7 days or saline control. After 10 days, thesizes of the open wounds in PPF1-treated animals was significantlyreduced compared to saline control. (**p<0.006 by unpaired T-test).

FIG. 25 reports the percentage of the wound still open at several timepoints post-wounding in a second similar study (Study 2). Mice weretreated with either PPF1 (150 μL) for 7 days or saline control. After 8days, the sizes of the open wounds in PPF1-treated animals wassignificantly reduced compared to saline control. (**p<0.0018 unpairedT-test).

FIG. 26 reports the percentage of the wound still open at 11 dayspost-wounding, combining the data from Studies 1 and 2. PPF1-treatedanimals exhibits a statistically-significant decrease in the percentageof wound left open after 11 days. (**p<0.006 by unpaired T-test). Thedifference between PPF1-treated and vehicle-treated animals at Day 10was similarly significant (**p<0.006 by unpaired T-test).

FIG. 27 reports the results of a study using topically-administered PPF1or vehicle to wounds in B6 ob/ob (B6.Cg-Lepob/J mice). FIG. 27 shows thestudy paradigm of daily administrations of 30 μL of topical PPF1 orcontrol vehicle administered to the wounds. Wounding was performed asdescribed in FIG. 10 .

FIG. 28 reports the results of the topical study, with percentage of thearea of the initial wound left after 10 days of treatment. FIG. 28indicates that PPF1 significantly decreased percentage of the open woundleft after 10 days compared to control vehicle.

DETAILED DESCRIPTION OF THE INVENTION A. Introduction

The present invention relates to the identification and discovery ofmethods and compositions for the treatment of unwanted conditionsassociated with postoperative recovery, and for improving such recovery.By “improving such recovery,” it is meant that a subject's postoperativerecovery may be accelerated, i.e. the subject may become mobile or bedischarged from in-patient care in less time than it would take withoutthe intervention of the embodiments of the present invention. By“unwanted conditions,” it is meant a condition or symptom such as, byway of example and not limitation, pain, cardiopulmonary issues,infections, thromboembolic issues, inflammation, and delayed woundhealing. Described herein are methods and compositions for the treatmentof subjects suffering from unwanted conditions associated withpostoperative recovery, and for improving such recovery, which areaspects of the present invention. Also described herein are dosingregimens which trigger improvement in subjects suffering from unwantedconditions associated with postoperative recovery, and for improvingsuch recovery. The methods and compositions described herein are usefulin: preventing complications from postoperative recovery; amelioratingthe symptoms of preventing complications from postoperative recovery;and accelerating postoperative recovery. The methods and compositions ofthe invention may be utilized or administered preoperatively (beforesurgery); perioperatively (during surgery); or postoperatively (aftersurgery).

Another aspect of the invention is for treating chronic pain/neuropathymore generally, and not exclusively chronic pain/neuropathy associatedwith postoperative recovery. The methods and compositions of theinvention described herein can be used to treat chronic pain andneuropathy. By “treating chronic pain and neuropathy” it is meant thatthe degree of chronic pain experienced by the subject to whom isadministered the compositions of the invention is lessened, slightly,moderately, or significantly as assessed by subjective or objectivemeans. Such means may include self- or medical professional-administeredtests such as, by way of example and not limitation: X-ray; MRI, CTscans; patient rating or description of the pain; range of motion;reflexes, muscle strength; sensitivity (e.g. how long it takes for thesubject to remove a limb that is subjected to pressure or otherstimulus); blood tests for inflammatory markers; electromyography (EMG);and nerve conduction velocity).

An implementation of the invention includes using blood plasma fractionsas treatment, such as one or more fractions or effluents obtained fromblood fractionation processes, e.g., like the Cohn fractionation processdescribed below. An embodiment of the invention includes using PlasmaFraction (a solution comprised of normal human albumin, alpha and betaglobulins, gamma globulin, and other proteins either individually or ascomplexes, hereinafter referred to as “Plasma Fraction”). Anotherembodiment of the invention includes using Plasma Protein Fraction (PPF)as treatment. Another embodiment of the invention includes using HumanAlbumin Solution (HAS) fraction as treatment. Yet another embodimentincludes using effluents from blood fractionation processes such asEffluent I or Effluent II/III described below. An additional embodimentincludes a blood plasma fraction from which substantially all theclotting factors have been removed in order to retain efficacy whilereducing the risk of thromboses (for example, see U.S. PatentApplication Nos. 62/236,710 and 63/376,529, which are incorporated byreference in their entirety herein).

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to a particular method orcomposition described, as such may, of course, vary. It is alsounderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

It is noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements or use of a“negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein have discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or the spirit of thepresent invention. Any recited method can be carried out in the order ofevents recited or in any other order which is logically possible.

B. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one having ordinaryskill in the art to which the invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, some potentialand preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those having skill in the art, and so forth.

In describing methods of the present invention, the terms “host”,“subject”, “individual” and “patient” are used interchangeably and referto any mammal in need of such treatment according to the disclosedmethods. Such mammals include, e.g., humans, ovines, bovines, equines,porcines, canines, felines, non-human primate, mice, and rats. Incertain embodiments, the subject is a non-human mammal. In someembodiments, the subject is a farm animal. In other embodiments, thesubject is a pet. In some embodiments, the subject is mammalian. Incertain instances, the subject is human. Other subjects can includedomestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats,horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g.,as in animal models of disease), as well as non-human primates (e.g.,chimpanzees, and monkeys). As such, subjects of the invention, includebut are not limited to mammals, e.g., humans and other primates, such aschimpanzees and other apes and monkey species; and the like, where incertain embodiments the subject are humans. The term subject is alsomeant to include a person or organism of any age, weight or otherphysical characteristic, where the subjects may be an adult, a child, aninfant or a newborn.

By a “young” or “young individual” it is meant an individual that is ofchronological age of 40 years old or younger, e.g., 35 years old oryounger, including 30 years old or younger, e.g., 25 years old oryounger or 22 years old or younger. In some instances, the individualthat serves as the source of the young plasma-comprising blood productis one that is 10 years old or younger, e.g., 5 years old or younger,including 1-year-old or younger. In some instances, the subject is anewborn and the source of the plasma product is the umbilical cord,where the plasma product is harvested from the umbilical cord of thenewborn. As such, “young” and “young individual” may refer to a subjectthat is between the ages of 0 and 40, e.g., 0, 1, 5, 10, 15, 20, 25, 30,35, or 40 years old. In other instances, “young” and “young individual”may refer to a biological (as opposed to chronological) age such as anindividual who has not exhibited the levels of inflammatory cytokines inthe plasma exhibited in comparatively older individuals. Conversely,these “young” and “young individual” may refer to a biological (asopposed to chronological) age such as an individual who exhibits greaterlevels of anti-inflammatory cytokines in the plasma compared to levelsin comparatively older individuals. By way of example, and notlimitation, the inflammatory cytokine is Eotaxin, and the folddifference between a young subject or young individual and olderindividuals is at least 1.5-fold. Similarly, the fold difference betweenolder and younger individuals in other inflammatory cytokines may beused to refer to a biological age. (See U.S. patent application Ser. No.13/575,437 which is herein incorporated by reference). Usually, theindividual is healthy, e.g., the individual has no hematologicalmalignancy or autoimmune disease at the time of harvest.

As used herein, “treatment” refers to any of (i) the prevention of thedisease or disorder, or (ii) the reduction or elimination of symptoms ofthe disease or disorder. Treatment may be effected prophylactically(prior to the onset of disease) or therapeutically (following the onsetof the disease). The effect may be prophylactic in terms of completelyor partially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. Thus, the term “treatment”as used herein covers any treatment of a condition associated withpostoperative recovery in a mammal and includes: (a) preventing thecondition from occurring in a subject; (b) inhibiting the condition,i.e., arresting its occurrence; or (c) relieving the condition, i.e.,causing regression of the condition. Treatment may result in a varietyof different physical manifestations, e.g., modulation in geneexpression, rejuvenation of tissue or organs, decreasing inflammation,etc. The therapeutic agent may be administered before, during or afterthe onset of the condition. The subject therapy may be administeredduring the symptomatic stage of the condition, and in some cases afterthe symptomatic stage of the condition.

Blood Products Comprising Plasma Components. In practicing the subjectmethods, a blood product comprising plasma components is administered toan individual in need thereof, e.g., an individual suffering from apostoperative condition. As such, methods according to embodiments ofthe invention include administering a blood product comprising plasmacomponents from an individual (the “donor individual”, or “donor”) to anindividual suffering from a postoperative condition (the “recipientindividual” or “recipient”). By a “blood product comprising plasmacomponents,” it is meant any product derived from blood that comprisesplasma (e.g. whole blood, blood plasma, or fractions thereof). The term“plasma” is used in its conventional sense to refer to thestraw-colored/pale-yellow liquid component of blood composed of about92% water, 7% proteins such as albumin, gamma globulin, anti-hemophilicfactor, and other clotting factors, and 1% mineral salts, sugars, fats,hormones and vitamins. Non-limiting examples of plasma-comprising bloodproducts suitable for use in the subject methods include whole bloodtreated with anti-coagulant (e.g., EDTA, citrate, oxalate, heparin,etc.), blood products produced by filtering whole blood to remove whiteblood cells (“leukoreduction”), blood products consisting ofplasmapheretically-derived or apheretically-derived plasma, fresh-frozenplasma, blood products consisting essentially of purified plasma, andblood products consisting essentially of plasma fractions. In someinstances, plasma product that is employed is a non-whole blood plasmaproduct, by which is meant that the product is not whole blood, suchthat it lacks one or more components found in whole blood, such aserythrocytes, leukocytes, etc., at least to the extent that thesecomponents are present in whole blood. In some instances, the plasmaproduct is substantially, if not completely, acellular, where in suchinstances the cellular content may be 5% by volume or less, such as 1%or less, including 0.5% or less, where in some instances acellularplasma fractions are those compositions that completely lack cells,i.e., they include no cells.

Collection of blood products comprising plasma components. Embodimentsof the methods described herein include administration of blood productscomprising plasma components which can be derived from donors, includinghuman volunteers. The term, “human-derived” can refer to such products.Methods of collection of plasma comprising blood products from donorsare well-known in the art. (See, e.g., AABB TECHNICAL MANUAL, (Mark A.Fung, et al., eds., 18th ed. 2014), herein incorporated by reference).

In one embodiment, donations are obtained by venipuncture. In anotherembodiment, the venipuncture is only a single venipuncture. In anotherembodiment, no saline volume replacement is employed. In a preferredembodiment, the process of plasmapheresis is used to obtain the plasmacomprising blood products. Plasmapheresis can comprise the removal of aweight-adjusted volume of plasma with the return of cellular componentsto the donor. In the preferred embodiment, sodium citrate is used duringplasmapheresis in order to prevent cell clotting. The volume of plasmacollected from a donor is preferably between 690 to 880 mL after citrateadministration, and preferably coordinates with the donor's weight.

C. Plasma Fractions

During the Second World War, there arose a need for a stable plasmaexpander which could be employed in the battlefield when soldiers lostlarge amounts of blood. As a result, methods of preparing freeze-driedplasma were developed. However, use of freeze-dried plasma was difficultin combat situations since reconstitution required sterile water. As analternative, Dr. E. J. Cohn suggested that albumin could be used, andprepared a ready-to-use stable solution that could be introducedimmediately for treatment of shock. (See Johan, Current Approaches tothe Preparation of Plasma Fractions in (Biotechnology of Blood) 165(Jack Goldstein ed., 1st ed. 1991)). Dr. Cohn's procedure of purifyingplasma fractions utilized cold ethanol for its denaturing effect andemploys changes in pH and temperature to achieve separation.

An embodiment of the methods described herein includes theadministration of plasma fractions to a subject. Fractionation is theprocess by which certain protein subsets are separated from plasma.Fractionation technology is known in the art and relies on stepsdeveloped by Cohn et al. during the 1940s. (E. Cohn, Preparation andproperties of serum and plasma proteins. IV. A system for the separationinto fractions of the protein and lipoprotein components of biologicaltissues and fluids. 68 J Am Chem Soc 459 (1946), herein incorporated byreference). Several steps are involved in this process, each stepinvolving specific ethanol concentrations as well as pH, temperature,and osmolality shifts which result in selective protein precipitation.Precipitates are also separated via centrifugation or precipitation. Theoriginal “Cohn fractionation process” involved separation of proteinsthrough precipitates into five fractions, designated fraction I,fraction II+III, fraction IV-1, fraction IV-4 and fraction V. Albuminwas the originally identified endpoint (fraction V) product of thisprocess. In accordance with embodiments of the invention, each fraction(or effluent from a prior separation step) contains or potentiallycontains therapeutically-useful protein fractions. (See Thierry Burnouf,Modern Plasma Fractionation, 21(2) Transfusion Medicine Reviews 101(2007); Adil Denizli, Plasma fractionation: conventional andchromatographic methods for albumin purification, 4 J. Biol. & Chem.315, (2011); and T. Brodniewicz-Proba, Human Plasma Fractionation andthe Impact of New Technologies on the Use and Quality of Plasma-derivedProducts, 5 Blood Reviews 245 (1991), and U.S. Pat. Nos. 3,869,431,5,110,907, 5,219,995, 7,531,513, and 8,772,461 which are hereinincorporated by reference). Adjustment of the above experimentalparameters can be made in order to obtain specific protein fractions.

More recently, fractionation has reached further complexity, and assuch, comprises additional embodiments of the invention. This recentincrease in complexity has occurred through: the introduction ofchromatography resulting in isolation of new proteins from existingfractions like cryoprecipitate, cryo-poor plasma, and Cohn fractions;increasing IgG recovery by integrating chromatography and the ethanolfractionation process; and viral reduction/inactivation/removal. (Id.)In order to capture proteins at physiological pH and ionic strength,anion-exchange chromatography can be utilized. This preserves functionalactivity of proteins and/or protein fractions. Heparin and monoclonalantibodies are also used in affinity chromatography. Additionally,fractionation using gel filtration, fraction by salt, and fractionationby polyethylene glycol are used. (Hosseini M Iran J Biotech, 14(4):213-20 (2016) herein incorporated by reference). One of ordinary skillin the art would recognize that the parameters and techniques describedabove may be adjusted to obtain specifically-desired plasmaprotein-containing fractions.

Blood plasma fractionation can also be ammonium sulfate-based. (See,e.g., Odunuga O O, Biochem Compounds, 1:3 (2013); Wingfield P T, CurrProtoc Protein Sci, Appx. 3 (2001), herein incorporated by reference).In addition to obtaining specific blood fractions, ammoniumsulfate-based fractionation has been employed to reduce abundantproteins from plasma. (Saha S, et al., J. Proteomics Bioinform, 5(8)(2012), herein incorporated by reference).

In an embodiment of the invention, blood plasma is fractionated in anindustrial setting. Frozen plasma is thawed at 1° C. to 4° C. Continuousrefrigerated centrifugation is applied to the thawed plasma andcryoprecipitate isolated. Recovered cryoprecipitate is frozen at −30° C.or lower and stored. The cryoprecipitate-poor (“cryo-poor”) plasma isimmediately processed for capture (via, for example, primarychromatography) of labile coagulation factors such as factor IX complexand its components as well as protease inhibitors such as antithrombinand C1 esterase inhibitor. Serial centrifugation and precipitateisolation can be applied in subsequent steps. Such techniques are knownto one of ordinary skill in the art and are described, for example, inU.S. Pat. Nos. 4,624,780, 5,219,995, 5,288,853, and U.S. patentapplication nos. 20140343255 and 20150343025, which disclosures areincorporated by reference in their entirety herein.

In an embodiment of the invention, the plasma fraction may comprise aplasma fraction containing a substantial concentration of albumin. Inanother embodiment of the invention, the plasma fraction may comprise aplasma fraction containing a substantial concentration of IgG orintravenous immune globulin (IGIV) (e.g. Gamunex-C®). In anotherembodiment of the invention the plasma fraction may comprise an IGIVplasma fraction, such as Gamunex-C® which has been substantiallydepleted of immune globulin (IgG) by methods well-known by one ofordinary skill in the art, such as for example, Protein A-mediateddepletion. (See Keshishian, H., et al., Multiplexed, QuantitativeWorkflow for Sensitive Biomarker Discovery in Plasma Yields NovelCandidates for Early Myocardial Injury, Molecular & Cellular Proteomics,14 at 2375-93 (2015)). In an additional embodiment, the blood plasmafraction may be one in which substantially all the clotting factors areremoved in order to retain the efficacy of the fraction with reducedrisk of thromboses. For example, the plasma fraction may be a plasmafraction as described in U.S. Patent No. 62/376,529 filed on Aug. 18,2016; the disclosure of which is incorporated by reference in itsentirety herein.

D. Albumin Products

To those having ordinary skill in the art, there are two generalcategories of Albumin Plasma Products (“APP”): plasma protein fraction(“PPF”) and human albumin solution (“HAS”). PPF is derived from aprocess with a higher yield than HAS but has a lower minimum albuminpurity than HAS (>83% for PPF and >95% for HAS). (Production of humanalbumin solution: a continually developing colloid, P. Matejtschuk etal., British J. of Anaesthesia 85(6): 887-95, at 888 (2000)). In someinstances, PPF has albumin purity of between 83% and 95% oralternatively 83% and 96%. The albumin purity can be determined byelectrophoresis or other quantifying assays such as, for example, bymass spectrometry. Additionally, some have noted that PPF has adisadvantage because of the presence of protein “contaminants” such asPKA. Id. As a consequence, PPF preparations have lost popularity asAlbumin Plasma Products, and have even been delisted from certaincountries' Pharmacopoeias. Id. Contrary to these concerns, the inventionmakes beneficial use of these “contaminants.” Besides α, β, and γglobulins, as well as the aforementioned PKA, the methods of theinvention utilize additional proteins or other factors within the“contaminants” that promote processes such as neurogenesis, neuronalcell survival, improved cognition or motor function and decreasedneuroinflammation.

Those of skill in the art will recognize that there are, or have been,several commercial sources of PPF (the “Commercial PPF Preparations.”)These include Plasma-Plex™ PPF (Armour Pharmaceutical Co., Tarrytown,N.Y.), Plasmanate™ PPF (Grifols, Clayton, N.C.), Plasmatein™ (AlphaTherapeutics, Los Angeles, Calif.), and Protenate™ PPF (Baxter Labs,Inc. Deerfield, Ill.). Those of skill in the art will also recognizethat there are, or have been, several commercial sources of HAS (the“Commercial HAS Preparations.”) These include Albuminar™ (CSL Behring),AlbuRx™ (CSL Behring), Albutein™ (Grifols, Clayton, N.C.), Buminate™(Baxatla, Inc., Bannockburn, Ill.), Flexbumin™ (Baxalta, Inc.,Bannockburn, Ill.), and Plasbumin™ (Grifols, Clayton, N.C.).

1. Plasma Protein Fraction (Human) (PPF)

According to the United States Food and Drug Administration (“FDA”),“Plasma Protein Fraction (Human),” or PPF, is the proper name of theproduct defined as “a sterile solution of protein composed of albuminand globulin, derived from human plasma.” (Code of Federal Regulations“CFR” 21 CFR 640.90 which is herein incorporated by reference). PPF'ssource material is plasma recovered from Whole Blood prepared asprescribed in 21 CFR 640.1-640.5 (incorporated by reference herein), orSource Plasma prepared as prescribed in 21 CFR 640.60-640.76(incorporated by reference herein).

PPF is tested to determine it meets the following standards as per 21CFR 640.92 (incorporated by reference herein):

(a) The final product shall be a 5.0+/−0.30 percent solution of protein;and

(b) The total protein in the final product shall consist of at least 83percent albumin, and no more than 17 percent globulins. No more than 1percent of the total protein shall be gamma globulin. The proteincomposition is determined by a method that has been approved for eachmanufacturer by the Director, Center for Biologics Evaluation andResearch, Food and Drug Administration.

As used herein, “Plasma Protein Fraction” or “PPF” refers to a sterilesolution of protein composed of albumin and globulin, derived from humanplasma, with an albumin content of at least 83% with no more than 17%globulins (including α1, α2, β, and γ globulins) and other plasmaproteins, and no more than 1% gamma globulin as determined byelectrophoresis. (Hink, J. H., Jr., et al., Preparation and Propertiesof a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174)(1957)). PPF can also refer to a solid form, which when suspended insolvent, has similar composition. The total globulin fraction can bedetermined through subtracting the albumin from the total protein.(Busher, J., Serum Albumin and Globulin, CLINICAL METHODS: THE HISTORY,PHYSICAL, AND LABORATORY EXAMINATIONS, Chapter 10, Walker H K, Hall W D,Hurst J D, eds. (1990)).

2. Albumin (Human) (HAS)

According to the FDA, “Albumin (Human)” (also referred to herein as“HAS”) is the proper name of the product defined as “sterile solution ofthe albumin derived from human plasma.” (Code of Federal Regulations“CFR” 21 CFR 640.80 which is herein incorporated by reference.) Thesource material for Albumin (Human) is plasma recovered from Whole Bloodprepared as prescribed in 21 CFR 640.1-640.5 (incorporated by referenceherein), or Source Plasma prepared as prescribed in 21 CFR 640.60-640.76(incorporated by reference herein). Other requirements for Albumin(Human) are listed in 21 CFR 640.80-640.84 (incorporated by referenceherein).

Albumin (Human) is tested to determine if it meets the followingstandards as per 21 CFR 640.82:

(a) Protein concentration. Final product shall conform to one of thefollowing concentrations: 4.0+/−0.25 percent; 5.0+/−0.30 percent;20.0+/−1.2 percent; and 25.0+/−1.5 percent solution of protein.

(b) Protein composition. At least 96 percent of the total protein in thefinal product shall be albumin, as determined by a method that has beenapproved for each manufacturer by the Director, Center for BiologicsEvaluation and Research, Food and Drug Administration.

As used herein, “Albumin (Human)” or “HAS” refers to a to a sterilesolution of protein composed of albumin and globulin, derived from humanplasma, with an albumin content of at least 95%, with no more than 5%globulins (including α1, α2, β, and γ globulins) and other plasmaproteins. HAS can also refer to a solid form, which when suspended insolvent, has similar composition. The total globulin fraction can bedetermined through subtracting the albumin from the total protein.

As can be recognized by one having ordinary skill in the art, PPF andHAS fractions can also be freeze-dried or in other solid form. Suchpreparations, with appropriate additives, can be used to make tablets,powders, granules, or capsules, for example. The solid form can beformulated into preparations for injection by dissolving, suspending oremulsifying them in an aqueous or non-aqueous solvent, such as vegetableor other similar oils, synthetic aliphatic acid glycerides, esters ofhigher aliphatic acids or propylene glycol; and if desired, withconventional additives such as solubilizers, isotonic agents, suspendingagents, emulsifying agents, stabilizers and preservatives.

E. Clotting Factor-Reduced Fractions

Another embodiment of the invention uses a blood plasma fraction fromwhich substantially all of the clotting factors are removed in order toretain the efficacy of the fraction with reduced risk of thromboses.Conveniently, the blood product can be derived from a young donor orpool of young donors and can be rendered devoid of IgM in order toprovide a young blood product that is ABO compatible. Currently, plasmathat is transfused is matched for ABO blood type, as the presence ofnaturally occurring antibodies to the A and B antigens can result intransfusion reactions. IgM appears to be responsible for transfusionreactions when patients are given plasma that is not ABO matched.Removal of IgM from blood products or fractions helps eliminatetransfusion reactions in subjects who are administered the bloodproducts and blood plasma fractions of the invention.

Accordingly, in one embodiment, the invention is directed to a method oftreating a subject suffering from an unwanted condition associated withpostoperative recovery. The method comprises: administering to thesubject a blood product or blood fraction derived from whole-blood froman individual or pool of individuals, wherein the blood product or bloodfraction is substantially devoid of (a) at least one clotting factorand/or (b) IgM. In some embodiments, the individual(s) from whom theblood product or blood fraction is derived are young individuals. Insome embodiments, the blood product is substantially devoid of at leastone clotting factor and IgM. In certain embodiments, the blood productis substantially devoid of fibrinogen (Factor I). In additionalembodiments, the blood product substantially lacks erythrocytes and/orleukocytes. In further embodiments, the blood product is substantiallyacellular. In other embodiments, the blood product is derived fromplasma. Such embodiments of the invention are further supported by U.S.Patent Application No. 62/376,529 filed on Aug. 18, 2016, which isincorporated by reference in its entirety herein.

F. Protein-Enriched Plasma Protein Products Treatment

Additional embodiments of the invention use plasma fractions withreduced albumin concentration compared to PPF, but with increasedamounts of globulins and other plasma proteins (what have been referredto by some as “contaminants”). The embodiments, as with PPF, HAS,Effluent I, and Effluent II/III are all effectively devoid of clottingfactors. Such plasma fractions are hereinafter referred to as“protein-enriched plasma protein products.” For example, an embodimentof the invention may use a protein-enriched plasma protein productcomprised of 82% albumin and 18% α, β, and γ globulins and other plasmaproteins. Another embodiment of the invention may use a protein-enrichedplasma protein product comprised of 81% albumin and 19% of α, β, and γglobulins and/or other plasma proteins. Another embodiment of theinvention may use a protein-enriched plasma protein product comprised of80% albumin and 20% of α, β, and γ globulins and/or other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 70-79% albumin anda corresponding 21-30% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 60-69% albumin anda corresponding 31-40% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 50-59% albumin anda corresponding 41-50% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 40-49% albumin anda corresponding 51-60% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 30-39% albumin anda corresponding 61-70% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 20-29% albumin anda corresponding 71-80% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 10-19% albumin anda corresponding 81-90% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 1-9% albumin and acorresponding 91-99% of α, β, and γ globulins and other plasma proteins.A further embodiment of the invention may use protein-enriched plasmaprotein products comprised of 0% albumin and 100% of α, β, and γglobulins and other plasma proteins

Embodiments of the invention described above may also have total gammaglobulin concentrations of 1-5%.

The specific concentrations of proteins in a plasma fraction may bedetermined using techniques well-known to a person having ordinary skillin the relevant art. By way of example, and not limitation, suchtechniques include electrophoresis, mass spectrometry, ELISA analysis,and Western blot analysis.

G. Preparation of Plasma Fractions

Methods of preparing PPF and other plasma fractions are well-known tothose having ordinary skill in the art. An embodiment of the inventionallows for blood used in the preparation of human plasma proteinfraction to be collected in flasks with citrate or anticoagulant citratedextrose solution (or other anticoagulant) for inhibition ofcoagulation, with further separation of Fractions I, II+III, IV, and PPFas per the method disclosed in Hink et al. (See Hink, J. H., Jr., etal., Preparation and Properties of a Heat-Treated Human Plasma ProteinFraction, VOX SANGUINIS 2(174) (1957), herein incorporated byreference.) According to this method, the mixture can be collected to2-8° C. The plasma can then subsequently be separated by centrifugationat 7° C., removed, and stored at −20° C. The plasma can then be thawedat 37° C. and fractionated, preferably within eight hours after removalfrom −20° C. storage.

Plasma can be separated from Fraction I using 8% ethanol at pH 7.2 and atemperature at −2 to −2.5° C. with protein concentration of 5.1 to 5.6percent. Cold 53.3 percent ethanol (176 mL/L of plasma) with acetatebuffer (200 mL 4M sodium acetate, 230 mL glacial acetic acid quantum satis to 1 L with H₂O) can be added using jets at a rate, for example, of450 mL/minute during the lowering the plasma temperature to −2° C.Fraction I can be separated and removed from the effluent (Effluent I)through ultracentrifugation. Fibrinogen can be obtained from Fraction Ias per methods well-known to those having ordinary skill in the art.

Fraction II+III can be separated from Effluent I through adjustment ofthe effluent to 21 percent ethanol at pH 6.8, temperature at −6° C.,with protein concentration of 4.3 percent. Cold 95 percent ethanol (176mL/L of Effluent I) with 10 M acetic acid used for pH adjustment can beadded using jets at a rate, for example, of 500 mL/minute during thelowering of the temperature of Effluent I to −6° C. The resultingprecipitate (Fraction II+III) can be removed by centrifugation at −6° C.Gamma globulin can be obtained from Fraction II+III using methodswell-known to those having ordinary skill in the art.

Fraction IV-1 can be separated from Effluent II+III (“Effluent II/III”)through adjustment of the effluent to 19 percent ethanol at pH 5.2,temperature at −6° C., and protein concentration of 3 percent. H₂O and10 M acetic acid used for pH adjustment can be added using jets whilemaintaining Effluent II/III at −6° C. for 6 hours. Precipitated FractionVI-1 can be settled at −6° C. for 6 hours and subsequently separatedfrom the effluent by centrifugation at the same temperature. Stableplasma protein fraction can be recovered from Effluent IV-1 throughadjustment of the ethanol concentration to 30 percent at pH 4.65,temperature −7° C. and protein concentration of 2.5 percent. This can beaccomplished by adjusting the pH of Effluent IV-1 with cold acid-alcohol(two parts 2 M acetic acid and one-part 95 percent ethanol). Whilemaintaining a temperature of −7° C., to every liter of adjusted EffluentIV-1 170 mL cold ethanol (95%) is added. Proteins that precipitate canbe allowed to settle for 36 hours and subsequently removed bycentrifugation at −7° C.

The recovered proteins (stable plasma protein fraction) can be dried(e.g. by freeze drying) to remove alcohol and H₂O. The resulting driedpowder can be dissolved in sterile distilled water, for example using 15liters of water/kg of powder, with the solution adjusted to pH 7.0 with1 M NaOH. A final concentration of 5 percent protein can be achieved byadding sterile distilled water containing sodium acetyl tryptophanate,sodium caprylate, and NaCl, adjusting to final concentrations of 0.004 Macetyl tryptophanate, 0.004 M caprylate, and 0.112 M sodium. Finally,the solution can be filtered at 10° C. to obtain a clear solution andsubsequently heat-treated for inactivation of pathogens at 60° C. for atleast 10 hours.

One having ordinary skill in the art would recognize that each of thedifferent fractions and effluents described above could be used with themethods of the invention to treat conditions associated withpostoperative recovery. For example, and not by way of limitation,Effluents I or Effluent II/III may be utilized to treat conditionsassociated with postoperative recovery or to accelerate postoperativerecovery and are embodiments of the invention.

The preceding methods of preparing plasma fractions and plasma proteinfraction (PPF) are only exemplary and involve merely embodiments of theinvention. One having ordinary skill in the art would recognize thatthese methods can vary. For example, pH, temperature, and ethanolconcentration, among other things can be adjusted to produce differentvariations of plasma fractions and plasma protein fraction in thedifferent embodiments and methods of the invention. In another example,additional embodiments of the invention contemplate the use ofnanofiltration for the removal/inactivation of pathogens from plasmafractions and plasma protein fraction.

An additional embodiment of the invention contemplates methods andcomposition using and/or comprising additional plasma fractions. Forexample, the invention, among other things, contemplates that specificconcentrations of albumin are not critical for treating conditionsassociated with postoperative recovery or for accelerating postoperativerecovery. Hence, fractions with reduced albumin concentration, such asthose fractions having below 83% albumin, are contemplated by theinvention.

H. Treatment

Aspects of the methods of the inventions described herein includetreatment of a subject with a plasma comprising blood product, such as ablood plasma fraction, e.g., as described above. An embodiment includestreatment of a human subject with a plasma comprising blood product. Oneof skill in the art would recognize that methods of treatment ofsubjects with plasma comprising blood products are recognized in theart. By way of example, and not limitation, one embodiment of themethods of the inventions described herein is comprised of administeringfresh frozen plasma to a subject for treatment of conditions associatedwith postoperative recovery. In one embodiment, the plasma comprisingblood product is administered immediately, e.g., within about 12-48hours of collection from a donor, to the individual suffering from acondition associated with postoperative recovery. In such instances, theproduct may be stored under refrigeration, e.g., 0-10° C. In anotherembodiment, fresh frozen plasma is one that has been stored frozen(cryopreserved) at −18° C. or colder. Prior to administration, the freshfrozen plasma is thawed and once thawed, administered to a subject 60-75minutes after the thawing process has begun. Each subject preferablyreceives a single unit of fresh frozen plasma (200-250 mL), the freshfrozen plasma preferably derived from donors of a pre-determined agerange. In one embodiment of the invention, the fresh frozen plasma isdonated by (derived from) young individuals. In another embodiment ofthe invention, the fresh frozen plasma is donated by (derived from)donors of the same gender. In another embodiment of the invention, thefresh frozen plasma is donated by (derived from) donors of the age rangebetween 18-22 years old.

In an embodiment of the invention the compositions of the invention thecompositions (e.g. plasma comprising blood product, such as a bloodplasma fraction) are administered intravenously. The compositions of theinvention may also be delivered intraperitoneally. In another embodimentof the invention, the compositions of the invention may be delivered peros, subcutaneously, or topically. Topical formulations for treatingwounds and promoting would healing as known in the art as gels, creams,ointments, gauze, patches and the like, and the compositions of theinvention may be formulated as such. (See, e.g., Kahn A W, et al.,Pharmacogn Mag, 9(Suppl 1):S6-S10 (2013); U.S. Pat. Nos. 5,641,483;4,885,163; 8,313,764, which are incorporated herein in their entirety).

In an embodiment of the invention, the plasma comprising blood productsare screened after donation by blood type. In another embodiment of theinvention, the plasma comprising blood products are screened forinfectious disease agents such as HIV I & II, HBV, HCV, HTLV I & II,anti-HBc per the requirements of 21 CFR 640.33 and recommendationscontained in FDA guidance documents.

In yet another embodiment of the invention, the subject is treated witha Plasma Fraction. In an embodiment of the invention, the plasmafraction is a PPF or a HAS. In a further embodiment of the invention,the plasma fraction is one of the Commercial PPF Preparations of theCommercial HAS Preparations. In another embodiment of the invention theplasma fraction is a PPF or HAS derived from a pool of individuals of aspecific age range, such as young individuals, or is a modified PPF orHAS fraction which has been subjected to additional fractionation orprocessing (e.g. PPF or HAS with one or more specific proteins partiallyor substantially removed). In another embodiment of the invention, theplasma fraction is an IGIV plasma fraction which has been substantiallydepleted of immune globulin (IgG). A blood fraction which is“substantially depleted” or which has specific proteins “substantiallyremoved,” such as IgG, refers to a blood fraction containing less thanabout 50% of the amount that occurs in the reference product or wholeblood plasma, such as less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 5%,4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, undetectable levels, or any integerbetween these values, as measured using standard assays well known inthe art.

I. Administration

Aspects of the methods of the inventions described herein includetreatment of a subject with a plasma comprising blood product, such as ablood plasma or Plasma Fraction, e.g., as described above. An embodimentincludes treatment of a human subject with a plasma comprising bloodproduct. One of skill in the art would recognize that methods oftreatment of subjects with plasma comprising blood products arerecognized in the art. By way of example, and not limitation, oneembodiment of the methods of the inventions described herein iscomprised of administering fresh frozen plasma to a subject fortreatment of conditions associated with postoperative recovery. In oneembodiment, the plasma comprising blood product is administeredimmediately, e.g., within about 12-48 hours of collection from a donor,to the individual suffering from an unwanted condition associated withpostoperative recovery. In such instances, the product may be storedunder refrigeration, e.g., 0-10° C. In another embodiment, fresh frozenplasma is one that has been stored frozen (cryopreserved) at −18° C. orcolder. Prior to administration, the fresh frozen plasma is thawed andonce thawed, administered to a subject 60-75 minutes after the thawingprocess has begun. Each subject preferably receives a single unit offresh frozen plasma (200-250 mL), the fresh frozen plasma preferablyderived from donors of a pre-determined age range. In one embodiment ofthe invention, the fresh frozen plasma is donated by (derived from)young individuals. In another embodiment of the invention, the freshfrozen plasma is donated by (derived from) donors of the same gender. Inanother embodiment of the invention, the fresh frozen plasma is donatedby (derived from) donors of the age range between 18-22 years old.

In an embodiment of the invention, the plasma comprising blood productsare screened after donation by blood type. In another embodiment of theinvention, the plasma comprising blood products are screened forinfectious disease agents such as HIV I & II, HBV, HCV, HTLV I & II,anti-HBc per the requirements of 21 CFR 640.33 and recommendationscontained in FDA guidance documents.

In yet another embodiment of the invention, the subject is treated witha Plasma Fraction. In an embodiment of the invention, the plasmafraction is PPF or HAS. In a further embodiment of the invention, theplasma fraction is one of the Commercial PPF Preparations or theCommercial HAS Preparations. In another embodiment of the invention theplasma fraction is a PPF or HAS derived from a pool of individuals of aspecific age range, such as young individuals, or is a modified PPF orHAS fraction which has been subjected to additional fractionation orprocessing (e.g. PPF or HAS with one or more specific proteins partiallyor substantially removed). In another embodiment of the invention, theplasma fraction is an IGIV plasma fraction which has been substantiallydepleted of immune globulin (IgG). A blood fraction which is“substantially depleted” or which has specific proteins “substantiallyremoved,” such as IgG, refers to a blood fraction containing less thanabout 50% of the amount that occurs in the reference product or wholeblood plasma, such as less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 5%,4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, undetectable levels, or any integerbetween these values, as measured using standard assays well known inthe art.

An embodiment of the invention includes treating a subject sufferingfrom a condition associated with postoperative recovery by administeringto the subject an effective amount of blood plasma or Plasma Fraction.Another embodiment of the invention includes administering the effectiveamount of blood plasma or Plasma Fraction and subsequently monitoringthe subject for improved function, wound healing, the presence ofmarkers, decreased pain, or decreased inflammation. Another embodimentof the invention includes treating a subject suffering from a conditionassociated with postoperative recovery by administering to the subjectan effective amount of blood plasma or Plasma Fraction wherein the bloodplasma or Plasma Fraction is administered in a manner resulting inimproved function wound healing, the presence of markers, decreasedpain, or decreased inflammation after the mean or median half-life ofthe blood plasma proteins or Plasma Fraction proteins been reached,relative to the most recent administered dose (referred to as “PulsedDosing” or “Pulse Dosed” herein) (See U.S. Pat. No. 10,357,513 and U.S.patent application Ser. Nos. 15/961,618 and 62/701,411, which are hereinincorporated by reference in their entirety). Another embodiment of theinvention includes administering the blood plasma or Plasma Fraction viaa dosing regimen of at least two consecutive days and monitoring thesubject for improved function or HSC marker levels at least 3 days afterthe date of last administration. A further embodiment of the inventionincludes administering the blood plasma or Plasma Fraction via a dosingregimen of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14consecutive days and monitoring the subject for improved function, woundhealing, the presence of markers, decreased pain, or decreasedinflammation at least 3 days after the date of last administration. Yetanother embodiment of the invention includes administering the bloodplasma or Plasma Fraction via a dosing regimen of at least 2 consecutivedays and after the date of last administration, monitoring forfunctional improvement, wound healing, the presence of markers,decreased pain, or decreased inflammation beyond when the averagehalf-life of the proteins in the blood plasma or Plasma Fraction hasbeen reached. Another embodiment of the invention includes administeringthe blood plasma or Plasma Fraction via a dosing regimen of 2 to 14non-consecutive days wherein each gap between doses may be between 0-3days each.

In some instances, Pulsed Dosing in accordance with the inventionincludes administration of a first set of doses, e.g., as describedabove, followed by a period of no dosing, e.g., a “dosing-free period”,which in turn is followed by administration of another dose or set ofdoses. The duration of this “dosing-free” period, may vary, but in someembodiments, is 7 days or longer, such as 10 days or longer, including14 days or longer, wherein some instances the dosing-free period rangesfrom 15 to 365 days, such as 30 to 90 days and including 30 to 60 days.As such, embodiments of the methods include non-chronic (i.e.,non-continuous) dosing, e.g., non-chronic administration of a bloodplasma product. In some embodiments, the pattern of Pulsed Dosingfollowed by a dosing-free period is repeated for a number of times, asdesired, where in some instances this pattern is continued for 1 year orlonger, such as 2 years or longer, up to and including the life of thesubject. Another embodiment of the invention includes administering theblood plasma or Plasma Fraction via a dosing regimen of 5 consecutivedays, with a dosing-free period of 2-3 days, followed by administrationfor 2-14 consecutive days.

Biochemically, by an “effective amount” or “effective dose” of activeagent is meant an amount of active agent that will inhibit, antagonize,decrease, reduce, or suppress by about 20% or more, e.g., by 30% ormore, by 40% or more, or by 50% or more, in some instances by 60% ormore, by 70% or more, by 80% or more, or by 90% or more, in some casesby about 100%, i.e., to negligible amounts, and in some instances,reverse unwanted conditions associated with postoperative recovery.

J. Plasma Protein Fraction

In practicing methods of the invention, a plasma fraction isadministered to the subject. In an embodiment, the plasma fraction isplasma protein fraction (PPF). In additional embodiments, the PPF isselected from the Commercial PPF Preparations.

In another embodiment, the PPF is comprised of 88% normal human albumin,12% alpha and beta globulins and not more than 1% gamma globulin asdetermined by electrophoresis. Further embodiments of this embodimentused in practicing methods of the invention include, for example, theembodiment as a 5% solution of PPF buffered with sodium carbonate andstabilized with 0.004 M sodium caprylate and 0.004 M acetyltryptophan.Additional formulations, including those modifying the percentage of PPF(e.g. about 1% to about 10%, about 10% to about 20%, about 20% to 25%,about 25% to 30%) in solution as well as the concentrations of solventand stabilizers may be utilized in practicing methods of the invention.

K. Plasma Fractions of Specific Donor Age

Additional embodiments of the invention include administering a plasmaprotein fraction derived from the plasma of individuals of certain ageranges. An embodiment includes administering PPF or HAS which have beenderived from the plasma of young individuals. In another embodiment ofthe invention the young individuals are of a single specific age or aspecific age range. In yet another embodiment, the average age of thedonors is less than that of the subject or less than the average age ofthe subjects being treated.

Certain embodiments of the invention include pooling blood or bloodplasma from individuals of specific age ranges and fractionating theblood plasma as described above to attain a plasma protein fractionproduct such as PPF or HAS. In an alternate embodiment of the invention,the plasma protein fraction or specific plasma protein fraction isattained from specific individuals fitting a specified age range.

L. Indications

The subject methods and plasma-comprising blood products and fractionsfind use in treating unwanted conditions associated with postoperativerecovery and even accelerating postoperative recovery. Such conditionsand indications include, by way of example and not limitation, pain andwound healing. The subject methods and compositions of the inventionalso find use in treating acute and chronic pain in diseases orconditions not necessarily related to postoperative recovery. Thesubject methods and compositions also find use in treating wound healingthat is not necessarily associated with postoperative recovery. Thesubject methods and compositions also find use in promoting orstimulating remyelination and treating diseases related to myelinationsuch as multiple sclerosis.

The subject methods and plasma-comprising blood products and fractionsalso find use in treating indications associated with the nervoussystem. Such conditions, by way of example and not limitation, includecentral nervous system conditions such as central neuropathic pain,spinal cord injury, myelopathy, and central neuropathic pain associatedwith postoperative recovery. Seventeen thousand new cases of spinalinjury occur per year with a prevalence of about 300,000, of which40-75% of subjects with spinal injury having central neuropathic pain.(Jadad A et al., AHRQ Evidence Report Summaries, Agency for HealthcareResearch and Quality; (1998-2005);https://www.nscisc.uab.edu/Public/Facts%202016.pdf; andhttps://www.nscisc.uab.edu/PublicDocuments/fact_figures_docs/Facts%202012%20Feb%20Final.pdf).One-third of patients experience intense pain with only ⅓ having a 50%or greater reduction in pain with treatment. (Charbonneau R, CMAJ,189(2):E48-E49 (2017); and Hadjipavlou G, et al., BJA Education,16(8):264-68 (2016)). Myelopathy has an occurrence rate of 605 per1,000,000 with surgical options, but no pharmacologic treatments,indicating an unmet need in the field. (Nouri A, et al., Spine,40(12):E675-93 (2015); The Lancet Neurology, editorial 18(7):P615(2019)).

These conditions also include, by way of example and not limitation,plexus/nerve root conditions such as plexopathy, cervical radiculopathy,and sciatica (lumbar radiculopathy). Plexopathy has a 2-3 per 100,000incidence. Its current options include management of neuropathic painwith antiepileptics and antidepressants, indicating an unmet need.Cervical radiculopathy's incidence is 100 per 100,000 males and 60 per100,000 females. (McCartney S, et al., Br. J. Gen. Pract., 68(666):44-46(2018)). Sciatica has an annual incidence of 1-5% and although manycases resolve spontaneously, sciatica becomes less responsive totreatment with prolonged duration of episodes. Treatments optionsinclude surgical procedures, standard pain medications, and steroids,indicating a need for new therapies. (Lewis R, et al., Health TechnologyAssessment—The Clinical Effectiveness and Cost-Effectiveness ofManagement Strategies for Sciatica: Systematic Review and EconomicModel, No. 15.39 NIHR Journals Library (2011)).

Additional indications include peripheral nervous system disorders.These include, by way of example and not limitation: peripheralneuropathy; peripheral neuropathy associated with postoperativerecovery; carpal tunnel syndrome; chemotherapy-induced peripheralneuropathy; compression and trauma; diabetic neuropathy; peripheralneuropathy associated with shingles (postherpetic neuralgia); complexregional pain syndrome; and trigeminal neuralgia. Peripheral neuropathyis a disorder of the peripheral nerves and affects at least 20 millionpeople in the United States along. Almost 60 percent of subjects withdiabetes experience diabetic neuropathy, a type of peripheralneuropathy.(http://www.healthcommunities.com/neuropathy/overview-of-neuropathy.shtml).Carpal tunnel syndrome affects 3-6% of adults, and treatments includesplints, steroids, and surgery. (LeBlanc K E, et al., Am Fam Physician,83(8):952-58 (2011)). Chemotherapy-induced peripheral neuropathy occursin 40-60% of patients both during and up to 3 months after receivingchemotherapy, with 650,000 patients reported to receive chemotherapy peryear. Peripheral neuropathy leads to dose reductions in chemotherapy oreven discontinuation, impacting quality of life, with no medication orsupplement having been shown to prevent the disorder. (JAMA Oncology,5(5):750, (2019)). Peripheral neuropathy related to compression andtrauma occurs in 2-3% of trauma patients, with 3 million cases of traumaoccurring in the United States. Although surgery is often effective,there is a need for new pharmacological agents. (American Associationfor the Surgery of Trauma—Trauma Facts, available athttp://www.aast.org/trauma-facts; and Novak C B, Medscape—PeripheralNerve Injuries, (Oct. 5, 2018) available athttps://emedicine.medscape.com/article/1270360-overview).

Further peripheral nervous system indications that the subject methodsand plasma-comprising blood products and fractions also find use intreating include diabetic neuropathy. In the United States, thepopulation of diabetes patients is about 30 million, and 8-26% of thosepatients suffer from neuropathy. (Risson V, et al., Incidence andprevalence of painful diabetic neuropathy and postherpetic neuralgia inmajor 5 European countries, the United States and Japan, Value in Health(20):A339-A811 PSY18 (2017), available athttps://www.valueinhealthjournal.com/article/S1098-3015(17)31179-8/pdf).The FDA-approved options for diabetic neuropathic pain includepregabalin, duloxetine, fluoxetine, and tapentadol, all of which manypatients do not respond to and none of which directly addresses nervedamage.

Peripheral neuropathy associated with shingles (postherpetic neuralgia)may also be treated by the methods and products of the invention. Twentypercent of shingles patients experience postherpetic neuralgia and thereare 1 million cases per year in the United States. (Seehttps://emedicine.medscape.com/article/1143066-overview#a6https://www.cdc.gov/shingles/hcp/clinical-overview.html.) Gabapentin andpregabalin are approved treatments for the condition but the pain isoften refractory to treatment. (Sacks G M, Am J Manag Care 19(1Suppl):S207-13 (2013)).

Additional peripheral neuropathic indications such as complex regionalpain syndrome and trigeminal neuralgia may be treated with the methodsand compositions of the invention. Five and one half to twenty-six casesoccur per 100,000 population. It is associated with severe pain anddisability and response to treatment is variable, indicating a highunmet need. (Complex Region Pain Syndrome Fact Sheet, NationalInstitutes of Health—National Institute of Neurological Disorders andStroke, available athttps://www.ninds.nih.gov/Disorders/Patient-Caregiver-Education/Fact-Sheets/Complex-Regional-Pain-Syndrome-Fact-Sheet).Trigeminal neuralgia occurs in 4.2-28.9 per 100,000 population. It has asignificant impact on quality of life, and can become resistant totreatment over time, requiring patients to try many differenttreatments. (Wu N, et al., J Pain, 18(Suppl 4):569, (2017)). The onlyapproved treatment is carbamazepine. Hence, there is an unmet need totreat the pain experienced by these patients.

Additional indications that may be treated with the methods andcompositions of the invention include the following examples: centralpost stroke pain; central pain in multiple sclerosis; post-traumaticheadaches; Dejerine-Roussy syndrome; optic neuritis; mitochondrial opticneuropathies; ischemic optic neuropathy; neuromyelitis optica;hereditary optic neuropathies; alcoholic neuropathy; Guillain-BarréSyndrome; Chronic Inflammatory Demyelinating Polyneuropathy (CIDP);Multifocal Motor Neuropathy (MNN); paraneoplastic autonomic neuropathy;peripheral neuropathy associated with sarcoidosis; peripheral neuropathyassociated with rheumatoid arthritis; peripheral neuropathy associatedwith systemic lupus erythematosus; peripheral neuropathy associated withSjögren's Syndrome; peripheral neuropathy associated with celiacdisease; Bell's palsy; peripheral neuropathy associated with Lymedisease; peripheral neuropathy associated with leprosy; peripheralNeuropathy associated with Hepatitis B; peripheral neuropathy associatedwith Hepatitis C; peripheral neuropathy associated with HIV/AIDS;peripheral neuropathy associated with amyloidosis; peripheral neuropathyassociated with anti-MAG; peripheral neuropathy associated withcryoglobulinemia; peripheral neuropathy associated with POEMS;toxin-Induced peripheral neuropathy; peripheral neuropathy associatedwith kidney disease; peripheral neuropathy associated with vasculitis;peripheral neuropathy associated with vitamin and nutrition deficiency;Charcot-Marie Tooth Disease (CMT); idiopathic peripheral neuropathy;fibromyalgia; and paraneoplastic peripheral neuropathy.

The subject methods and plasma-comprising blood products and fractionsalso find use in treating indications associated with wound healing.Wounds may be, for example and not as limitation, abrasions, avulsions,incisions, lacerations, and punctures. Such indications can include bothchronic wounds and acute wounds. By way of example, and not limitation,wound indications include: chronic wounds such as diabetic ulcer;pressure ulcer; venous ulcer; arterial ulcer; as well as acute woundssuch as surgical wounds; traumatic wounds; and burns. But any type ofchronic or acute wound may be treated by the subject methods andcompositions of the invention.

Diabetic ulcers affect over 2.2 million people in the United States witha global incidence of 6.4%. (Chun D, et al., J Clin Med, 8:748 (2019)).Despite several treatment options such as debridement and medicaldressings, many patients endure infection and eventually requireamputation, highlighting the need for new remedies, in particularpharmacological remedies.

Pressure ulcers occur at an overall rate of 1.8% of hospital admittees,with the total number of annual cases being in the hundreds ofthousands. (Bauer K, et al., Ostomy Wound Manage, 62(11):30-38 (2016)).Like diabetic ulcers, treatment options such as debridement and medicaldressing exist, but many patients experience infection and the ulcerscan lead to mortality.

Venous ulcers occur primarily in the leg and comprise a substantialburden on the elderly and occur in about 1% of populations worldwide.(Nelzen O, Phlebolymphology, 15(4) (2008)). Venous ulcers are difficultto heal and have a significant tendency to recur than other chroniculcers. As with diabetic and pressure ulcers, treatment options such asdebridement and medical dressing exist, but their recurrence highlightsa need for new treatments, in particularly pharmacological-basedtreatments. Arterial ulcers occur at a rate of approximately a quarterof the rate of venous ulcers. (Gabriel A, Vascular Ulcers, (2018),available athttps://emedicine.medscape.com/article/1298345-overview#a6). Treatmentoptions also include debridement and medical dressings, but there is alack of approved pharmacological agents.

Surgical wounds occur in approximately 1.3 million patients per year.(See MediWound—Innovating Solutions for Wound & Burn Care (2019) at 19available athttp://ir.mediwound.com/static-files/cd547017-d1ed-460e-8cb2-0550b1e18a29).Surgical wounds are cuts or incisions in the skin usually made by ascalpel during surgery but can also result from a drain placed duringsurgery. Healing of surgical wounds is a critical outcome for surgery.Postoperative wound disruption or separation of the layers of the woundwith fascial disruption can be a serious complication. (See HospitalHarm Improvement Resource—Wound Disruption (2016), available athttps://www.patientsafetyinstitute.ca/en/toolsResources/Hospital-Hann-Measure/Documents/Resource-Library/HHIR%20Wound%20Disruption.pdf).Additionally, healing of surgical wounds takes considerably more time inelderly patients compared to younger individuals. (Gerstein A D,Dermatol Clin, 11(4):749-57 (1993).

Traumatic wounds are primarily cuts, lacerations, puncture, or abrasionwounds with damage having been caused to the skin and the underlyingtissues. Traumatic wounds are typically classified under three groups:acute wounds; cut wounds, and penetrating wounds. Acute wounds are whenthe skin is ripped or torn, the wound's appearance is jagged, andusually contain foreign bodies like glass, metal, gravel, sand or dirt.Cut wounds are when a sharp object penetrates the skin and underlyingsubcutaneous tissues. Penetrating wounds are the deepest of the threetypes and the most severe. Stab wounds and gunshot wounds are typicalexamples. (See Traumatic Wounds available athttps://www.woundcarecenters.org/article/wound-types/traumatic-wounds;and Leaper D J, BMJ, 332(7540):532-35 (2006)). Although there areseveral physical treatment options (e.g. sutures), there remains a needfor pharmacological interventions.

The World Health Organization estimates that 180,000 deaths occur everyyear as a result of burns. And non-fatal burn injuries are a leadingcause of morbidity, including prolonged hospitalization.(https://www.who.int/news-room/fact-sheets/detail/burns). Typicaltreatment includes surgical management and dressings. Pharmacologicaltreatment is focused on analgesia, infection control, sedation,circulating blood volume replacement, anticoagulation, and nutrition.(Green A, et al., Clinical Pharmacist, 2:249-54 (2010)). The methods andcompositions of the invention can fill an unmet need for pharmacologicalintervention that promotes healing of the damage to the skin andunderlying tissues.

The subject methods and plasma-comprising blood products and fractionscan be used to treat conditions and indications associated withpostoperative recovery at different time points. For example, and not asa limitation, administration to a subject can be performed:pre-operatively, perioperatively (during the procedure), orpost-operatively.

One embodiment of the invention is that the subject methods andplasma-comprising blood products and fractions can be used to treatpain. Such pain, by way of example and not limitation, may include acuteor chronic pain. Another embodiment of the invention is that the subjectmethods and plasma-comprising blood products and fractions can also beused to treat central pain or central neuropathy. Central pain includesneurological conditions caused by damage to or dysfunction of thecentral nervous system (CNS), including the brain, brainstem, and spinalcord. It may affect a large portion of the body or it can be restrictedto specific areas. The pain may be constant or intermittent. The painmay be moderate to severe in intensity. Such pain may also be affectedby touch, movement, emotions, and temperature changes. The pain may alsohave an immediate onset after the causative incident or may be delayedby months or years. (See Central Pain Information Page—NationalInstitute of Neurological Disorders and Stroke, Central Pain SyndromeInformation Page, available athttps://www.ninds.nih.gov/disorders/all-disorders/central-pain-syndrome-information-page;and Colloca L, et al., Nat Rev Dis Primers, 3:17002 (2017)). Furtherembodiments of the invention include using the subject methods andplasma-comprising blood productions and fractions to treat: spinal cordinjury (SCI); myelopathy; plexopathy; cervical radiculopathy; sciatica(lumbar radiculopathy); central post stroke pain; central pain inmultiple sclerosis; post-traumatic headaches; Dejerine-Roussy syndrome;optic neuritis; mitochondrial optic neuropathies; ischemic opticneuropathy; neuromyelitis optica; and hereditary optic neuropathies.

Another embodiment of the invention is that the subject methods andplasma-comprising blood products and fractions can also be used to treatperipheral pain or peripheral neuropathy. Peripheral neuropathy canrefer to several conditions involving damage to the peripheral nervoussystem. More than 100 peripheral neuropathies have been identified anddepend on what type(s) of nerve(s) is/are damaged including motornerves, sensory nerves, and autonomic nerves. (See Central PageInformation Page—National Institute of Neurological Disorders andStroke, Peripheral Neuropathy Fact Sheet, available athttps://www.ninds.nih.gov/Disorders/Patient-Caregiver-Education/Fact-Sheets/Peripheral-Neuropathy-Fact-Sheet;and Colloca L, et al., Nat Rev Dis Primers, 3:17002 (2017)). Furtherembodiments of the invention include using the subject methods andplasma-comprising blood productions and fractions to treat: carpaltunnel syndrome; chemo-induced peripheral neuropathy; compression andtrauma; diabetic neuropathy; peripheral neuropathy associated withShingles (postherpetic neuralgia); complex regional pain syndrome;trigeminal neuralgia; alcoholic neuropathy; Guillain-Barré Syndrome;Chronic Inflammatory Demyelinating Polyneuropathy (CIDP); MultifocalMotor Neuropathy (MNN); paraneoplastic autonomic neuropathy; peripheralneuropathy associated with sarcoidosis; peripheral neuropathy associatedwith rheumatoid arthritis; peripheral neuropathy associated withsystemic lupus erythematosus; peripheral neuropathy associated withSjögren's Syndrome; peripheral neuropathy associated with celiacdisease; Bell's palsy; peripheral neuropathy associated with Lymedisease; peripheral neuropathy associated with leprosy; peripheralneuropathy associated with Hepatitis B; peripheral neuropathy associatedwith Hepatitis C; peripheral neuropathy associated with HIV/AIDS;peripheral neuropathy associated with amyloidosis; peripheral neuropathyassociated with anti-MAG; peripheral neuropathy associated withcryoglobulinemia; peripheral neuropathy associated with POEMS;Toxin-Induced peripheral neuropathy; peripheral neuropathy associatedwith kidney disease; peripheral neuropathy associated with vasculitis;peripheral neuropathy associated with vitamin and nutrition deficiency;Charcot-Marie Tooth Disease (CMT); idiopathic peripheral neuropathy;fibromyalgia; and paraneoplastic peripheral neuropathy.

One embodiment of the invention is that the subject methods andplasma-comprising blood products and fractions can be used to treatwounds by promoting wound healing. Further embodiments of the inventioninclude using the subject methods and plasma-comprising bloodproductions and fractions to treat chronic or acute wounds. Additionalembodiments of the invention include treating: diabetic ulcers; pressureulcers; venous ulcers; arterial ulcers; surgical wounds; traumaticwounds; and burns.

M. Reagents, Devices, and Kits

Also provided are reagents, devices, and kits thereof for practicing oneor more of the above-described methods. The subject reagents, devices,and kits thereof may vary greatly.

Reagents and devices of interest include those mentioned above withrespect to the methods of preparing plasma-comprising blood product fortransfusion into a subject in need hereof, for example, anti-coagulants,cryopreservatives, buffers, isotonic solutions, etc.

Kits may also comprise blood collection bags, tubing, needles,centrifugation tubes, and the like. In yet other embodiments, kits asdescribed herein include two or more containers of blood plasma productsuch as plasma protein fraction, such as three or more, four or more,five or more, including six or more containers of blood plasma product.In some instances, the number of distinct containers of blood plasmaproduct in the kit may be 9 or more, 12 or more, 15 or more, 18 or more,21 or more, 24 or more 30 or more, including 36 or more, e.g., 48 ormore. Each container may have associated therewith identifyinginformation which includes various data about the blood plasma productcontained therein, which identifying information may include one or moreof the age of the donor of the blood plasma product, processing detailsregarding the blood plasma product, e.g., whether the plasma product wasprocessed to remove proteins above an average molecule weight (such asdescribed above), blood type details, etc. In some instances, eachcontainer in the kit includes identifying information about the bloodplasma contained therein, and the identifying information includesinformation about the donor age of the blood plasma product, e.g., theidentifying information provides confirming age-related data of theblood plasma product donor (where such identifying information may bethe age of the donor at the time of harvest). In some instances, eachcontainer of the kit contains a blood plasma product from a donor ofsubstantially the same age, i.e., all of the containers include productfrom donors that are substantially the same, if not the same, age. Bysubstantially the same age is meant that the various donors from whichthe blood plasma products of the kits are obtained differ in each, insome instances, by 5 years or less, such as 4 years or less, e.g., 3years or less, including 2 years or less, such as 1 year or less, e.g.,9 months or less, 6 months or less, 3 months or less, including 1 monthor less. The identifying information can be present on any convenientcomponent of the container, such as a label, an RFID chip, etc. Theidentifying information may be human readable, computer readable, etc.,as desired. The containers may have any convenient configuration. Whilethe volume of the containers may vary, in some instances the volumesrange from 10 ml to 5000 mL, such as 25 mL to 2500 mL, e.g., 50 ml to1000 mL, including 100 mL to 500 mL. The containers may be rigid orflexible, and may be fabricated from any convenient material, e.g.,polymeric materials, including medical grade plastic materials. In someinstances, the containers have a bag or pouch configuration. In additionto the containers, such kits may further include administration devices,e.g., as described above. The components of such kits may be provided inany suitable packaging, e.g., a box or analogous structure, configuredto hold the containers and other kit components.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, portable flash drive, etc., on which the informationhas been recorded. Yet another means that may be present is a websiteaddress which may be used via the internet to access the information ata removed site. Any convenient means may be present in the kits.

N. Experimental Examples

1. Models for Pain

a) Pain—Treatment Before Injury

(1) Alteration of Neuropathic Nerve Injury

A chronic pain model employing chronic constrictive injury (CCI) wasused to determine levels of pain experienced by 22-month-old C57BL/6Jmice treated with: (1) PPF1 following CCI; (2) vehicle following CCI; or(3) vehicle following sham surgery. Using such a model, the nervoussystem becomes regulated to a persistent state of high reactivity whichlowers the pain threshold long after the initial injury has occurred.(See, e.g., Safakhah, H. A. et. al., Journal of Pain, 10:1457-66 andSuter M R, et al., Anesthesiology Res and Practice (2011) which areherein incorporated by reference in their entirety).

PPF1 is a PPF with approximately 88% normal human albumin (in relationto total protein), 12% alpha and beta globulins, and no more than 1%gamma globulin as determined by electrophoresis. Except where noted,PPF1 is administered in the examples herein in vivo using a 5% solution(w/v, 50 g/L). PPF2 is also a PPF, but a different lot from PPF1. PPF2meets the same protein content and concentration specifications as PPF1.

FIG. 1 depicts timeline of a CCI experiment. Twenty-three-month-old wildtype mice were administered a CCI or sham surgery via ligation 24 hoursprior to administration of a 7-consecutive-day pulse dosing regimen of150 uL/day (intravenously tail-vein) of either PPF1 or vehicle control.Behavior was assessed during week four, and tissue collection forhistology occurred at week five.

FIG. 2 is a representation depicting the location of the CCIadministered to twenty-three-month-old wild type mice. The ligation wasadministered on the sciatic nerve as indicated by the figure. The figurewas adapted from Suter M R, et al., Anesthesiology Res and Practice,(2011), which is incorporated herein by reference in its entirety.

FIG. 3 reports data from a mechanical von Frey allodynia test inwild-type mice 4 weeks after CCI or sham surgery as detailed in FIG. 1 .To determine an animal's tolerance to mechanical pressure, the hind pawenervated by the subject sciatic nerve, was stimulated by differingthicknesses of von Frey filaments. The pressure at which the mousewithdrew its hind paw was measured and plotted in FIG. 3 . The figureillustrates that mice treated with PPF1 after CCI exhibitedsignificantly less pain (could withstand more pressure) than thosetreated with vehicle control after CCI. Sham surgery animals alsoexhibited significantly less pain that those treated with vehiclecontrol after CCI. The primary finding is that PPF1 has a positiveeffect on mechanical nociception deficits induced by CCI. ***P<0.001 CCItreated with PPF1 vs. CCI Vehicle treatment, *P<0.05 Sham vehicle vs.CCI vehicle; One-way ANOVA with Tukey post-hoc analysis.

FIG. 4 reports data from hippocampal histology performed on the wildtype mice described in FIG. 1 . Neurogenesis was measured using thedoublecortin (DCX) marker. Mice who received CCI surgery and weretreated with PPF1 had significantly increased neurogenesis in thedentate gyrus of the hippocampus than those who received vehicle. Micewho received sham operation trended towards greater neurogenesis thanmice who received CCI surgery, both groups received vehicle treatmentpost-surgery. Thus, PPF1 exhibited the ability to restore neurogenesisafter chronic nerve injury. *P<0.05 CCI treated with PPF1 vs. CCIVehicle treatment; Unpaired T-Test.

FIG. 5 reports data from hippocampal histology performed on the wildtype mice described in FIG. 1 . Inflammatory marker as measured by CD68expression was quantified. Our findings illustrate that mice whichreceived CCI surgery and vehicle treatment expressed a significantlygreater number of CD68 positive cells in the hippocampus than those weretreated with PPF1 following CCI surgery. PPF1 treated animals hadsimilar inflammation levels to that of the sham surgery group. Thisillustrates that PPF1 can help to ameliorate neuroinflammation resultingfrom chronic nerve injury. *P<0.05 CCI treated with PPF1 vs. CCI Vehicletreatment, Sham vehicle vs. CCI vehicle; One-way ANOVA with Tukeypost-hoc analysis.

FIG. 6 reports data from a mechanical von Frey allodynia test inC57BL/6J mice which received CCI or sham surgery and tested in atimeline as described in FIG. 1 . Twenty-two-month-old mice wereadministered a 7-consecutive-day pulse dosing regimen of 150 uL/day(intravenous tail-vein) of either PPF1 or vehicle control. Another groupreceived Gabapentin at 75 mg/kg (intraperitoneal administration) dailyfor 7 consecutive days. All treatments were initiated 24 hours after CCIor sham surgery. To determine an animal's tolerance to mechanicalpressure, the hind paw enervated by the subject sciatic nerve, wasstimulated by differing thicknesses of von Frey filaments. The pressureat which the mouse withdrew its hind paw was assessed and represented inFIG. 6 as weeks post CCI or sham surgery. The figure illustrates thatmice administered PPF1 following CCI surgery had significantly increasedtolerance to mechanical nociception at all assessed timepoints thanthose treated with vehicle after CCI. Conversely, mice administeredGabapentin only show significant improvement in mechanical nociceptionat 2 weeks following CCI surgery and are similar to vehicle treated miceat all other timepoints. Sham surgery mice show significantly increasedresponse to mechanical nociception at 3 and 5 weeks following surgicalmanipulation. Together, these data illustrate that PPF1 amelioratesperipheral pain for a greater amount of time than that of standard ofcare treatments (Gabapentin). ***, ****P<0.001, P<0.0001 PPF1 vs.Vehicle control; ANOVA with Tukey Post-hoc analysis. *P<0.05 Gabapentinvs. Vehicle control; ANOVA with Tukey Post-hoc analysis. *, ** P<0.05,P<0.01 Sham vs. Vehicle control; ANOVA with Tukey Post-hoc analysis.

FIG. 7 reports data from a hot plate test on wild-type mice treated asdescribed in FIG. 1 and as described by Woolfe and Macdonald. (Woolfe G.and Macdonald A D, J. Pharmacol. Exp. Ther. 80:300-07 (1944), which isincorporated by reference herein in its entirety). The hot plate is setto a temperature of 55° C. Mice are acclimated to being placed inside aclear cylinder for 30 minutes. The cylinder is placed upon the hot plateand a timer started. When nocifensive behaviors (e.g. hind paw lickingor jumping) are first observed, the time is recorded as latency. If nonocifensive behaviors are observed, the animal is removed at apre-determined cut-off time such as 30 seconds to prevent tissue damage.Mice are only tested at 2- and 5-weeks post CCI surgery, as repetitiveexposure to testing has been shown to alter sensitivity. FIG. 7illustrates hot plate nocifensive latency 5 weeks after CCI or shamsurgery. PPF1 treatment are significantly less sensitive to hot platestimuli compared to mice given CCI plus vehicle control, indicating arescue effect by PPF1. **P<0.01 Sham vs. CCI surgery, ****P<0.0001 PPF1vs. Vehicle treated CCI surgery mice. ANOVA with Tukey Post-hocanalysis.

(2) Prevention of Neuroinflammation in the Spinal Cord

A separate study similar to the preceding study (above) was performed on22-month-old C57BL/6J mice. Cohorts of mice were treated as follows: (1)PPF (PPF2) following CCI; (2) vehicle following CCI; (3) recombinanthuman albumin (rhAlb) following CCI; or (4) vehicle following shamsurgery. Mice were administered a 7-consecutive-day pulse dosing regimenof 150 μL/day (intravenous tail-vein) of PPF2, recombinant humanalbumin, or vehicle control. All treatments were initiated 24 hoursafter CCI or sham surgery.

FIG. 8 reports data from a hot plate test (as described above)thirty-five (35) days post CCI as treated in the timeline of FIG. 1 .PPF2-treated mice were significantly less sensitive to hot plate stimulicompared to mice given CCI plus vehicle control. Mice treated withrecombinant human albumin were also significantly less sensitive to micegiven CCI plus vehicle control, but not to the degree of mice treatedwith PPF2. *P<0.05 rhAlb vs. vehicle treated CCI mice, *** P<0.001 PPF2vs. vehicle treated CCI surgery mice. ANOVA with Tukey Post-hocanalysis.

FIG. 9 reports data from a mechanical von Frey allodynia test in thesesame mice at different time intervals both pre- (baseline) and post-CCI.The pressure at which the mouse withdrew their hind paws was assessedand is represented in FIG. 9 as weeks post CCI or sham surgery. Thefigure illustrates the mice administered PPF2 following CCI surgery hadsignificantly increased tolerance to mechanical nociception at allassessed timepoints than those treated with vehicle or recombinant humanalbumin (rhAlb) after CCI. This shows that a PPF (PPF2) ameliorated painfor a greater amount of time than control vehicle or albumin, albuminbeing the major protein component of PPF. Thus, these effects appear notto be mediated via albumin, but to other proteins present in PPF.*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 vs. vehicle control; ANOVAwith Tukey Post-hoc analysis.

FIG. 10 reports the relative levels of myelin basic protein (MBP,detected by Abcam, ab40390 anti-rabbit antibody) in the distal sciaticnerve five weeks after the last dose of PPF (PPF1) in another similarexperiment conducted in 22-month-old mice as described above. * P<0.05;***P<0.001 vs. vehicle control; ANOVA with Tukey Post-hoc analysis.

FIG. 11 reports the relative levels in these mice of S-100 Schwann cellmarker. In both cases, PPF in mice with CCI increased relative levels ofthese markers compared to vehicle control mice with CCI. Together thisshows that PPF promotes sciatic nerve repair mechanisms via increasingmyelin protein and S-100 protein expression. It also shows that PPFinduces myelination repair mechanisms. **P<0.01; ***P<0.001 vs. vehiclecontrol; ANOVA with Tukey Post-hoc analysis.

FIG. 12 is a fluorescence microscopic qualitative representation of thedata reported in FIGS. 10 and 11 .

FIG. 13 and FIG. 14 show detection of BDNF and CD68, respectively, inthe dorsal horn of the spinal cord in mice treated 24 hours post-CCIinjury. Brain-derived neurotrophic factor (BDNF, detected by Abcam,ab108319 anti-rabbit antibody) is secreted by activated microglia and ithas been shown to enhance spinal nociception (detection of painfulstimuli) through synaptic facilitation and engagement of centralsensitization-like mechanisms. Peripheral injury-induced neuropathicpain is often accompanied with increased spinal expression of BDNF(Garraway S M, et al. Neural Plast. Article ID 9857201 (2016)). CD68levels (detected by Biorad MCA1957 GA anti-rat antibody) were alsodetermined. CD68 is a marker for activated microglia. FIGS. 13 and 14show that PPF treatment 24 hours after CCI injury results in significantreduction of both BDNF and CD68 markers in the dorsal horns of thespinal cord, indicating the prevention of microglial activation andblocking of deleterious downstream events linked to development ofneuropathic pain. **P<0.01; ***P<0.001 vs. vehicle control; ANOVA withTukey Post-hoc analysis.

FIGS. 15 and 16 are fluorescent microscopic images of the data presentedin FIGS. 13 and 14 , respectively. The rectangle highlights the dorsalhorns of the spinal cord which was analyzed at the L4-L6 lumbar spinalsegments. The images on the right sides of the figures are higher focalpowered images of the rectangular regions on the left sides of eachfigure.

b) Pain—Treatment Fourteen Days after Injury

FIG. 17 shows the protocol used on 22-month-old C57BL/6J mice. Baselinevon Frey paw withdrawal thresholds for measuring mechanical allodyniawere taken 3-4 days before CCI or sham procedures. Cohorts of mice weretreated as follows: (1) PPF (PPF1) 14 days following CCI; (2) vehicle 14days following CCI; (3) recombinant human albumin (rhAlb) 14 daysfollowing CCI; or (4) vehicle 14 days following sham surgery. Mice wereadministered a 7-consecutive-day pulse dosing regimen of 150 μL/day(intravenous tail-vein) of PPF1, recombinant human albumin, or vehiclecontrol. All treatments were initiated 14 days after CCI or shamsurgery.

FIG. 18 reports the Von Frey paw withdrawal thresholds at baseline, 14,21, 28, 35, 42, and 49 days post-CCI. At Day 14, a significant deficitis seen in all but the sham group, indicating that there is centralsensitization in all CCI groups after 2 weeks of injury. This is notreversed until 7 days after cessation of treatment with PPF (Day 28),indicating that simple analgesia does not take place with PPF in thismodel. Instead, a mechanistic effect takes place with PPF treatmentwhich is not observed with vehicle or recombinant human albumin (rhAlbumin). This shows that pain that is fully established before PPFtreatment (which necessarily involves a central component) issignificantly alleviated by PPF compared to vehicle control. **P<0.01;*** P<0.001; ****P<0.0001 vs. vehicle control; ANOVA with Tukey Post-hocanalysis.

FIGS. 19 and 20 report the hot plate latency values at 35 Days post-CCI(FIG. 19 ) and 49 Days post-CCI (FIG. 20 ). Both sets of results showthat the PPF-treated mice had long-lasting reductions of hot plate painsensitivity. This also supports the observation that PPF works through amechanistic effect as opposed to simply providing an analgesic effect.**P<0.01; ANOVA with Tukey Post-hoc analysis.

2. Model for Wound Healing

A mouse model of diabetes (B6.BKS(D)-Lepr^(db)/J) was used to assess theefficacy of PPF1 on wound healing. Six-week-old male B6.BKS(D)-Leprdb/Jmice were shaved on their back on day −1. Mice were wounded on Day 0 ontwo locations on their back. Mice received daily treatments (IV) ofvehicle (150 μL) or PPF1 (150 μL) from day 0 (immediately after skinwounding) to 6 included.

Skin wounding was performed as follows: Animals were depilated the daybefore skin wounding using a depilating cream followed by gentle wash oftheir skin with warm water. Animals were anesthetized using inhalantisoflurane, surgical site was shaved and prepped with povidone-iodine(“betadine”) or chlorhexidine antiseptic products (or similar surgicalscrub) and 70% ethanol. Hot water heat pad (or similar surgical product)was placed underneath the mice. Two wounding sites were marked on theirdorsal skin with 5 mm-diameter circles using a permanent marker. Thedorsal skin was lifted using clean forceps and cut using fine surgicalscissors alongside the marked circles. A 15 mm-diameter silicone splintwith a 6 mm-diameter cut in its middle was applied around the woundusing Vetbond and nylon suture thread. After skin wounding, mice wereweighed (initial body weight) and placed in a clean home cage with aheating pad underneath and softened food. Mice stayed on pad and weremonitored, until mice were Bright, Alert, and Reactive.

Postoperatively, wound healing was assessed daily until sutures wereremoved. Buprenorphine was administered i.p. right after surgery andevery ˜12 hours for a total of three injections. Meloxicam wasadministered i.p. prior to surgery and 24 hours post-surgery. Softenedfood and Clear H₂O Recovery gel will be placed on the bottom of the cageafter surgery. Mouse body weight was measured daily post-surgery. If amouse lost more than 1 gram body weight since post-surgery weight, 500ul saline/day was administered.

Mice were evaluated daily for their amount of wound closure by measuringthe wound size using a caliper. Mice were sacrificed on Days 10 and 14.Mice were deeply anesthetized with Avertin (250 mg/kg IP) and thensubjected to cardiac puncture and blood samples collected withpre-filled syringes with EDTA. Blood/EDTA was then injected into amicrocentrifuge tube. The tubes were kept on ice and plasma separated bycentrifugation at 1000 g (+4° C.) for 15 min as soon as was possible.Plasma from each mouse is aliquoted at 100 μL per vial, with theremainder in a second vial, and stored at −80° C.

Skin was collected from each mouse, fixed and 4% paraformaldehydefollowed by 2 washes in PBS and subsequently paraffin embedded. Tissuesare sectioned or lysed and analyzed for markers of inflammation bystandard histological and biochemical methods, including qRT-PCR,Western blot, ELISA, and immunohistochemistry.

FIG. 21 is a histological comparison between a diabetic wound(B6.BKS(D)-Lepr^(db)/J diabetic mouse model) that was untreated (FIG.21A) or with PPF1 (FIG. 21B). Black bars indicate wound bed thickness(epidermal plus granulation layer). Arrows indicate wound boundaries.Wound bed thickness was increased in PPF1-treated mice as determined bywound bed thickness. PPF1 therefore demonstrates improved wound healing.

FIG. 22 is a histological comparison between a diabetic wound(B6.BKS(D)-Lepr^(db)/J diabetic mouse model) that was untreated (FIG.22A) or with PPF1 (FIG. 22B). Black bars indicate the granulation layer.Blue bars indicate the epidermal layer. The PPF1-treated wound exhibiteda thicker epidermal layer than the untreated wound, however thegranulation layer exhibited an even greater trend in the differencebetween PPF1-treated and untreated wounds (i.e. the granulation layerwas thicker in the PPF1-treated wounds than the untreated wounds).

FIG. 23 through FIG. 26 report results from the B6 ob/ob (B6.Cg-Lepob/Jmice) diabetic mouse model assessing the efficacy of PPF1 versus vehicleon diabetic wound healing. Nine-week-old male B6 OB/OB mice were used.The day before wounding, mice were weighed and fasted for 5 hours todetermine fasting glucose from tail blood. The mice were equally dividedinto 2 different treatment groups according to weight and glucose level.For wounding, mice were shaved, applied with hair removal cream (Nair™),wounded on two locations on their back (5 mm diameter excision), thenapplied with a silicone ring soaked in 70% alcohol (12 mm outercircumference and 6 mm inner circumference). The ring was attached tothe open wound with Vectabond™. Four sutures were applied to each ringto ensure that the silicone ring stayed attached to the wound throughthe entire course of the experiment. 30 μL of PPF1 and control wereapplied directly on top of the wound and the two wounds sealed with apiece of Tegaderm™. Daily treatments were administered by injecting PPF1and control inside the Tegaderm™ that covered the wounds. To image thewounds, the Tegaderm™ that was just covering the wound was cut off andthe wound resealed with a fresh piece of Tegaderm™. At sacrifice, therings were removed, and the wounds were cut from the back for histology.

Wound healing was assessed daily by determining the amount of woundclosure. Wound size was measured using a camera and a precision rulerfor scale. Terminal tissue collection was performed on Days 10 and 14.Tissues were sectioned or lysed and analyzed for markers of inflammationby standard histological methods including immunohistochemistry,qRT-PCR, and H&E and other special stains.

FIG. 23 depicts the general design of the experiment. Blood dropsindicate when blood was collected to measure fasting glucose level. OnDay 2, the skin wound was made, and on Days 1-7 intravenous (iv) dosingwas performed. FIG. 24 reports the percentage of the wound still open atseveral time points post-wounding in a first study (Study 1). Mice weretreated with either PPF1 (150 μL) for 7 days or saline control. After 10days, the sizes of the open wounds in PPF1-treated animals wassignificantly reduced compared to saline control. (**p<0.006 by unpairedT-test).

FIG. 25 reports the percentage of the wound still open at several timepoints post-wounding in a second similar study (Study 2). Mice weretreated with either PPF1 (150 μL) for 7 days or saline control. After 8days, the sizes of the open wounds in PPF1-treated animals wassignificantly reduced compared to saline control. (**p<0.0018 unpairedT-test).

FIG. 26 reports the percentage of the wound still open at 11 dayspost-wounding, combining the data from Studies 1 and 2. PPF1-treatedanimals exhibits a statistically-significant decrease in the percentageof wound left open after 11 days. (**p<0.006 by unpaired T-test). Thedifference between PPF1-treated and vehicle-treated animals at Day 10was similarly significant (**p<0.006 by unpaired T-test).

FIG. 27 and FIG. 28 report the results of a study usingtopically-administered PPF1 or vehicle to wounds in B6 ob/ob(B6.Cg-Lepob/J mice). FIG. 27 shows the study paradigm of dailyadministrations of 30 μL of topical PPF1 or control vehicle administeredto the wounds. Wounding was performed as described in FIG. 23 . FIG. 28reports the results of the topical study, with percentage of the area ofthe initial wound left after 10 days of treatment. FIG. 28 indicatesthat PPF1 significantly decreased percentage of the open wound leftafter 10 days compared to control vehicle.

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a limitation in the claim onlywhen the exact phrase “means for” or the exact phrase “step for” isrecited at the beginning of such limitation in the claim; if such exactphrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

What is claimed:
 1. A method of treating a subject diagnosed with pain,the method comprising administering an effective amount of a PlasmaFraction to the subject to treat the subject for pain.
 2. The method ofclaim 1 wherein the pain is chronic pain.
 3. The method of claim 1wherein the pain is peripheral pain.
 4. The method of claim 1 whereinthe pain is central pain.
 5. The method of claim 1 wherein the PlasmaFraction is a Plasma Protein Fraction.
 6. The method of claim 1 whereinthe Plasma Protein Fraction comprises between 83% to 95% albumin.
 7. Themethod of claim 6 wherein the Plasma Protein Fraction is a commerciallyavailable Plasma Protein Fraction.
 8. The method of claim 1 wherein theadministering is performed using a Pulse Dose dosing regimen.